Nanotoxicity Evaluation Methods

Nanomaterials are utilized across a wide spectrum of scientific disciplines, encompassing biomedicine among others. Though nanomaterials hold a great impact on the advancement of conventional systems, the toxicological implications of such nano-formulations must not be overlooked. Hence, an assessment of the toxicity profile of such compounds must be carried out in addition to their application-specific evaluation for animals as well as human well-being. The extent of nanotoxicity induction depends on their size, shape, nature, area/volume ratio, functionalization, and other physico chemical properties. Animals including human exposure to nano-fabrications may give rise to cytotoxicity leading to apoptosis and necrosis, generation of Reactive Oxygen Species (ROS) and oxidative stresses, abnormality in gene expression causing genotoxicity, immunological disturbances, etc. Several in-vivo as well as in-vitro methods for assessing nanotoxicity potential are required for the fruitful application of nanomaterials with minimal or zero side- effects. For instance, colorimetric assays such as MTT assay, LDH assay, etc. can be used for the determination of cell viability. TUNEL assay, COMET assay, Caspase assay, Annexin V assay, CLSM, FACS, and such other methods are involved in detection of apoptosis and necrosis. Along with DCFH assay and plasmid assay, determination of Glutathione level using diagnostic kits or microscopic observation is also carried out for evaluating ROS production. Comet assay, Ames test, etc. are performed for determining the potential of DNA damage, mutagenicity, and cell proliferation by NPs. Effects of nanomaterials on immunological health can be elicited through diverse in-vitro technologies including EpiScreen™, Epibase®, REVEAL®, and so on. The psychological impact of NPs can also be determined through several behavioral studies such as hole-board test (HBT), Elevated Plus Maze (EPM) test, Porsolt swim test (PST) as well as the hot plate test and the tail flick test. Toxic effects of NPs at the organ-tissue level are evaluated by histopathological tests using fluorescence microscopy. Furthermore, several Omics-based platforms such as Single-molecule real-time (SMRT) sequencing by using a droplet-based (Drop-Seq) system, DBiT-seq, single-cell genome long-read sequencing technology (SMOOTH-seq) along with, Mass Cytometry, Fluorescence Flow Cytometry, Enzyme-Linked Immunospot Assay, Single-Molecule Array (SiMoA), Microfluidic Antibody Capture Chip (MACS Chip)-based assay, Proximity Extension Assay/Abseq, are also exploited for toxicological studies. However, every method has its limitations as a single modality can depict the toxicity effect to an extent only. Hence, a comprehensive assessment including diverse models as well as modalities would be beneficial. Further, short as well as long-term toxicity impacts need to be investigated to get a wholesome depiction. Thus, the following chapter will enlighten the applications of different state-of-art modalities regarding the determination of nanotoxicity while focusing on animal health, including humans.